Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1996 Oct; 16(10): 5288–5301.
PMCID: PMC231528

Glucocorticoid-stimulated CCAAT/enhancer-binding protein alpha expression is required for steroid-induced G1 cell cycle arrest of minimal-deviation rat hepatoma cells.


By genetic correlation with the growth-suppressible phenotype and direct functional tests, we demonstrate that the glucocorticoid-stimulated expression of the CCAAT/enhancer-binding protein alpha (C/EBP alpha) transcription factor is required for the steroid-mediated G1 cell cycle arrest of minimal-deviation rat hepatoma cells. Comparison of C/EBP alpha transcript and active protein levels induced by the synthetic glucocorticoid dexamethasone in glucocorticoid growth-suppressible (BDS1), nonsuppressible receptor-positive (EDR1) and nonsuppressible receptor-deficient (EDR3) hepatoma cell proliferative variants revealed that the stimulation of C/EBP alpha expression is a rapid, glucocorticoid receptor-mediated response associated with the G1 cell cycle arrest. Consistent with the role of C/EBP alpha as a critical intermediate in the growth suppression response, maximal induction of transcription factor mRNA occurred within 2 h of dexamethasone treatment whereas maximal inhibition of [3H] thymidine incorporation was observed 24 h after steroid treatment. As a direct functional approach, ablation of C/EBP alpha protein expression and DNA-binding activity by transfection of an antisense C/EBP alpha expression vector blocked the dexamethasone-induced G1 cell cycle arrest of hepatoma cells but did not alter general glucocorticoid responsiveness. Transforming growth factor beta induced a G1 cell cycle arrest in C/EBP alpha antisense transfected cells, demonstrating the specific involvement of C/EBP alpha in the glucocorticoid growth suppression response. Constitutive expression of a conditionally activated form of C/EBP alpha caused a G1 cell cycle arrest of BDS1 hepatoma cells in the absence of glucocorticoids. In contrast, overexpression of C/EBP beta or C/EBP delta had no effect on hepatoma cell growth. Taken together, these results demonstrate that the steroid-induced expression of C/EBP alpha is necessary to mediate the glucocorticoid G1 cell cycle arrest of rat hepatoma cells and implicates a role for this transcription factor in the growth control of liver-derived epithelial tumor cells.

Full Text

The Full Text of this article is available as a PDF (726K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. [PMC free article] [PubMed]
  • Alam T, An MR, Mifflin RC, Hsieh CC, Ge X, Papaconstantinou J. trans-activation of the alpha 1-acid glycoprotein gene acute phase responsive element by multiple isoforms of C/EBP and glucocorticoid receptor. J Biol Chem. 1993 Jul 25;268(21):15681–15688. [PubMed]
  • Baumann H. Transcriptional control of the rat alpha 1-acid glycoprotein gene. J Biol Chem. 1990 Nov 15;265(32):19420–19423. [PubMed]
  • Baumann H, Gauldie J. Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation. Mol Biol Med. 1990 Apr;7(2):147–159. [PubMed]
  • Baumann H, Jahreis GP, Morella KK, Won KA, Pruitt SC, Jones VE, Prowse KR. Transcriptional regulation through cytokine and glucocorticoid response elements of rat acute phase plasma protein genes by C/EBP and JunB. J Biol Chem. 1991 Oct 25;266(30):20390–20399. [PubMed]
  • Baumann H, Maquat LE. Localization of DNA sequences involved in dexamethasone-dependent expression of the rat alpha 1-acid glycoprotein gene. Mol Cell Biol. 1986 Jul;6(7):2551–2561. [PMC free article] [PubMed]
  • Baumann H, Morella KK, Campos SP, Cao Z, Jahreis GP. Role of CAAT-enhancer binding protein isoforms in the cytokine regulation of acute-phase plasma protein genes. J Biol Chem. 1992 Sep 25;267(27):19744–19751. [PubMed]
  • Baumann H, Prowse KR, Marinković S, Won KA, Jahreis GP. Stimulation of hepatic acute phase response by cytokines and glucocorticoids. Ann N Y Acad Sci. 1989;557:280–296. [PubMed]
  • Beato M. Transcriptional control by nuclear receptors. FASEB J. 1991 Apr;5(7):2044–2051. [PubMed]
  • Beato M, Herrlich P, Schütz G. Steroid hormone receptors: many actors in search of a plot. Cell. 1995 Dec 15;83(6):851–857. [PubMed]
  • Birkenmeier EH, Gwynn B, Howard S, Jerry J, Gordon JI, Landschulz WH, McKnight SL. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 1989 Aug;3(8):1146–1156. [PubMed]
  • Buck M, Turler H, Chojkier M. LAP (NF-IL-6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation. EMBO J. 1994 Feb 15;13(4):851–860. [PMC free article] [PubMed]
  • Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. [PubMed]
  • Chang CJ, Chen TT, Lei HY, Chen DS, Lee SC. Molecular cloning of a transcription factor, AGP/EBP, that belongs to members of the C/EBP family. Mol Cell Biol. 1990 Dec;10(12):6642–6653. [PMC free article] [PubMed]
  • Christy RJ, Yang VW, Ntambi JM, Geiman DE, Landschulz WH, Friedman AD, Nakabeppu Y, Kelly TJ, Lane MD. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev. 1989 Sep;3(9):1323–1335. [PubMed]
  • Cook PW, Swanson KT, Edwards CP, Firestone GL. Glucocorticoid receptor-dependent inhibition of cellular proliferation in dexamethasone-resistant and hypersensitive rat hepatoma cell variants. Mol Cell Biol. 1988 Apr;8(4):1449–1459. [PMC free article] [PubMed]
  • Cross M, Dexter TM. Growth factors in development, transformation, and tumorigenesis. Cell. 1991 Jan 25;64(2):271–280. [PubMed]
  • Descombes P, Chojkier M, Lichtsteiner S, Falvey E, Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 1990 Sep;4(9):1541–1551. [PubMed]
  • Descombes P, Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991 Nov 1;67(3):569–579. [PubMed]
  • Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR. Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science. 1990 Sep 14;249(4974):1266–1272. [PubMed]
  • Diehl AM, Johns DC, Yang S, Lin H, Yin M, Matelis LA, Lawrence JH. Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes. J Biol Chem. 1996 Mar 29;271(13):7343–7350. [PubMed]
  • Diehl AM, Michaelson P, Yang SQ. Selective induction of CCAAT/enhancer binding protein isoforms occurs during rat liver development. Gastroenterology. 1994 Jun;106(6):1625–1637. [PubMed]
  • Diehl AM, Yang SQ. Regenerative changes in C/EBP alpha and C/EBP beta expression modulate binding to the C/EBP site in the c-fos promoter. Hepatology. 1994 Feb;19(2):447–456. [PubMed]
  • Draetta GF. Mammalian G1 cyclins. Curr Opin Cell Biol. 1994 Dec;6(6):842–846. [PubMed]
  • Elledge SJ, Harper JW. Cdk inhibitors: on the threshold of checkpoints and development. Curr Opin Cell Biol. 1994 Dec;6(6):847–852. [PubMed]
  • Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. [PubMed]
  • Fausto N, Laird AD, Webber EM. Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J. 1995 Dec;9(15):1527–1536. [PubMed]
  • Flodby P, Antonson P, Barlow C, Blanck A, Porsch-Hällström I, Xanthopoulos KG. Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats. Exp Cell Res. 1993 Sep;208(1):248–256. [PubMed]
  • Friedman AD, Landschulz WH, McKnight SL. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. [PubMed]
  • Fuller PJ. The steroid receptor superfamily: mechanisms of diversity. FASEB J. 1991 Dec;5(15):3092–3099. [PubMed]
  • Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. [PMC free article] [PubMed]
  • Goya L, Alexander DB, Webster MK, Kern FG, Guzman RC, Nandi S, Firestone GL. Overexpression of transforming growth factor alpha overrides the glucocorticoid-mediated suppression of Con8 mammary tumor cell growth in vitro and in vivo. Cancer Res. 1993 Apr 15;53(8):1816–1822. [PubMed]
  • Goya L, Edwards CP, Glennemeier KA, Firestone GL. In vivo effects of dexamethasone on the tumor growth of glucocorticoid-sensitive Fu5-derived rat hepatoma cells. Cancer Lett. 1991 Jul 4;58(3):211–219. [PubMed]
  • Goya L, Maiyar AC, Ge Y, Firestone GL. Glucocorticoids induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that is synchronously reversed by steroid withdrawal or addition of transforming growth factor-alpha. Mol Endocrinol. 1993 Sep;7(9):1121–1132. [PubMed]
  • Gronemeyer H. Control of transcription activation by steroid hormone receptors. FASEB J. 1992 May;6(8):2524–2529. [PubMed]
  • Groner B, Altiok S, Meier V. Hormonal regulation of transcription factor activity in mammary epithelial cells. Mol Cell Endocrinol. 1994 Apr;100(1-2):109–114. [PubMed]
  • Gunning P, Ponte P, Okayama H, Engel J, Blau H, Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol. 1983 May;3(5):787–795. [PMC free article] [PubMed]
  • Gustafsson JA, Carlstedt-Duke J, Poellinger L, Okret S, Wikström AC, Brönnegård M, Gillner M, Dong Y, Fuxe K, Cintra A. Biochemistry, molecular biology, and physiology of the glucocorticoid receptor. Endocr Rev. 1987 May;8(2):185–234. [PubMed]
  • Haber BA, Mohn KL, Diamond RH, Taub R. Induction patterns of 70 genes during nine days after hepatectomy define the temporal course of liver regeneration. J Clin Invest. 1993 Apr;91(4):1319–1326. [PMC free article] [PubMed]
  • Hartwell LH, Kastan MB. Cell cycle control and cancer. Science. 1994 Dec 16;266(5192):1821–1828. [PubMed]
  • Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. [PubMed]
  • Hattori M, Abraham LJ, Northemann W, Fey GH. Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2364–2368. [PMC free article] [PubMed]
  • Hendricks-Taylor LR, Darlington GJ. Inhibition of cell proliferation by C/EBP alpha occurs in many cell types, does not require the presence of p53 or Rb, and is not affected by large T-antigen. Nucleic Acids Res. 1995 Nov 25;23(22):4726–4733. [PMC free article] [PubMed]
  • Hunter T, Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994 Nov 18;79(4):573–582. [PubMed]
  • Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990 Sep 21;62(6):1189–1204. [PubMed]
  • Kinoshita S, Akira S, Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1473–1476. [PMC free article] [PubMed]
  • Kirschner M. The cell cycle then and now. Trends Biochem Sci. 1992 Aug;17(8):281–285. [PubMed]
  • Klein ES, DiLorenzo D, Posseckert G, Beato M, Ringold GM. Sequences downstream of the glucocorticoid regulatory element mediate cycloheximide inhibition of steroid induced expression from the rat alpha 1-acid glycoprotein promoter: evidence for a labile transcription factor. Mol Endocrinol. 1988 Dec;2(12):1343–1351. [PubMed]
  • Landschulz WH, Johnson PF, Adashi EY, Graves BJ, McKnight SL. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. [PubMed]
  • Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. [PubMed]
  • Leffert HL, Koch KS, Moran T, Rubalcava B. Hormonal control of rat liver regeneration. Gastroenterology. 1979 Jun;76(6):1470–1482. [PubMed]
  • Lewin B. Driving the cell cycle: M phase kinase, its partners, and substrates. Cell. 1990 Jun 1;61(5):743–752. [PubMed]
  • Lin FT, Lane MD. Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. Genes Dev. 1992 Apr;6(4):533–544. [PubMed]
  • Lin FT, Lane MD. CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8757–8761. [PMC free article] [PubMed]
  • Lin FT, MacDougald OA, Diehl AM, Lane MD. A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9606–9610. [PMC free article] [PubMed]
  • Lucas PC, Granner DK. Hormone response domains in gene transcription. Annu Rev Biochem. 1992;61:1131–1173. [PubMed]
  • MacDougald OA, Cornelius P, Lin FT, Chen SS, Lane MD. Glucocorticoids reciprocally regulate expression of the CCAAT/enhancer-binding protein alpha and delta genes in 3T3-L1 adipocytes and white adipose tissue. J Biol Chem. 1994 Jul 22;269(29):19041–19047. [PubMed]
  • Mischoulon D, Rana B, Bucher NL, Farmer SR. Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture. Mol Cell Biol. 1992 Jun;12(6):2553–2560. [PMC free article] [PubMed]
  • Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto S, Kasahara T, Matsushima K. Novel mechanism of glucocorticoid-mediated gene repression. Nuclear factor-kappa B is target for glucocorticoid-mediated interleukin 8 gene repression. J Biol Chem. 1994 May 6;269(18):13289–13295. [PubMed]
  • Natsuka S, Isshiki H, Akira S, Kishimoto T. Augmentation of haptoglobin production in Hep3B cell line by a nuclear factor NF-IL6. FEBS Lett. 1991 Oct 7;291(1):58–62. [PubMed]
  • Nerlov C, Ziff EB. Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-alpha on the serum albumin promoter. Genes Dev. 1994 Feb 1;8(3):350–362. [PubMed]
  • Nishio Y, Isshiki H, Kishimoto T, Akira S. A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction. Mol Cell Biol. 1993 Mar;13(3):1854–1862. [PMC free article] [PubMed]
  • Osada S, Yamamoto H, Nishihara T, Imagawa M. DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family. J Biol Chem. 1996 Feb 16;271(7):3891–3896. [PubMed]
  • Ossipow V, Descombes P, Schibler U. CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8219–8223. [PMC free article] [PubMed]
  • Pabo CO, Sauer RT. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. [PubMed]
  • Pardee AB. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. [PubMed]
  • Parker MG. Steroid and related receptors. Curr Opin Cell Biol. 1993 Jun;5(3):499–504. [PubMed]
  • Poli V, Mancini FP, Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990 Nov 2;63(3):643–653. [PubMed]
  • Rana B, Mischoulon D, Xie Y, Bucher NL, Farmer SR. Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. Mol Cell Biol. 1994 Sep;14(9):5858–5869. [PMC free article] [PubMed]
  • Rana B, Xie Y, Mischoulon D, Bucher NL, Farmer SR. The DNA binding activity of C/EBP transcription factor is regulated in the G1 phase of the hepatocyte cell cycle. J Biol Chem. 1995 Jul 28;270(30):18123–18132. [PubMed]
  • Ratajczak T, Williams PM, DiLorenzo D, Ringold GM. Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP. J Biol Chem. 1992 Jun 5;267(16):11111–11119. [PubMed]
  • Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):752–756. [PMC free article] [PubMed]
  • Rhee K, Reisman D, Bresnahan W, Thompson EA. Glucocorticoid regulation of G1 cyclin-dependent kinase genes in lymphoid cells. Cell Growth Differ. 1995 Jun;6(6):691–698. [PubMed]
  • Sánchez I, Goya L, Vallerga AK, Firestone GL. Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression. Cell Growth Differ. 1993 Mar;4(3):215–225. [PubMed]
  • Truss M, Beato M. Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. Endocr Rev. 1993 Aug;14(4):459–479. [PubMed]
  • Umek RM, Friedman AD, McKnight SL. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991 Jan 18;251(4991):288–292. [PubMed]
  • Wahli W, Martinez E. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 1991 Jun;5(9):2243–2249. [PubMed]
  • Watkins PJ, Condreay JP, Huber BE, Jacobs SJ, Adams DJ. Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein. Cancer Res. 1996 Mar 1;56(5):1063–1067. [PubMed]
  • Watts CK, Brady A, Sarcevic B, deFazio A, Musgrove EA, Sutherland RL. Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation. Mol Endocrinol. 1995 Dec;9(12):1804–1813. [PubMed]
  • Williams P, Ratajczak T, Lee SC, Ringold GM. AGP/EBP(LAP) expressed in rat hepatoma cells interacts with multiple promoter sites and is necessary for maximal glucocorticoid induction of the rat alpha-1 acid glycoprotein gene. Mol Cell Biol. 1991 Oct;11(10):4959–4965. [PMC free article] [PubMed]
  • Williams SC, Cantwell CA, Johnson PF. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 1991 Sep;5(9):1553–1567. [PubMed]
  • Xanthopoulos KG, Mirkovitch J, Decker T, Kuo CF, Darnell JE., Jr Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4117–4121. [PMC free article] [PubMed]
  • Yamamoto KR. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet. 1985;19:209–252. [PubMed]
  • Yeh WC, Cao Z, Classon M, McKnight SL. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev. 1995 Jan 15;9(2):168–181. [PubMed]
  • Zilliacus J, Wright AP, Carlstedt-Duke J, Gustafsson JA. Structural determinants of DNA-binding specificity by steroid receptors. Mol Endocrinol. 1995 Apr;9(4):389–400. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...